Discovery of a novel tetrahydroimidazo[1,2-a]pyridine-5-carboxylic acid derivative as a potent and selective heparanase-1 inhibitor utilizing an improved synthetic approach.

Heparanase-1 (HPSE1) is an endo-ß-d-glucuronidase that catalyzes degradation of heparan sulfate proteoglycans. Inhibition of HPSE1 appears to be a useful therapeutic target against cancer and proteinuric kidney diseases. We previously reported tetrahydroimidazo[1,2-a]pyridine 2 as a potent HPSE1 inhibitor after optimization of the synthetic reaction. However, synthesis of 2 involves a total of 19 steps, including a cyclization process that accompanies a strong odor due to the use of Lawesson's reagent and an epimerization reaction; furthermore, 2 exhibited insufficient selectivity for HPSE1 over exo-ß-d-glucuronidase (GUSß) and glucocerebrosidase (GBA), which also needed to be addressed. First, the cyclization reaction was optimized to synthesize tetrahydroimidazo[1,2-a]pyridine without using Lawesson's reagent or epimerization, with reference to previous reports. Next, 16 and 17 containing a bulkier substituent at position 6 than the 6-methoxyl group in 2 were designed and synthesized using the improved cyclization conditions, so that the synthetic route of 16 and 17 was shortened by five steps as compared with that of 2. The inhibitory activities of 16 and 17 against GUSß and GBA were reduced as compared with those of 2, that is, the compounds showed improved selectivity for HPSE1 over GUSß and GBA. In addition, 16 showed enhanced inhibitory activity against HPSE1 as compared with that of 2. Compound 16 appears promising as an HPSE1 inhibitor with therapeutic potential due to its highly potent inhibitory activity against HPSE1 with high selectivity for HPSE1.

Synthetic Heparanase Inhibitors Can Prevent Herpes Simplex Viral Spread.

Herpes simplex virus (HSV-1) employs heparan sulfate (HS) as receptor for cell attachment and entry. During late-stage infection, the virus induces the upregulation of human heparanase (Hpse) to remove cell surface HS allowing viral spread. We hypothesized that inhibition of Hpse will prevent viral release thereby representing a new therapeutic strategy for HSV-1. A range of HS-oligosaccharides was prepared to examine the importance of chain length and 2-O-sulfation of iduronic moieties for Hpse inhibition. It was found that hexa- and octasaccharides potently inhibited the enzyme and that 2-O-sulfation of iduronic acid is tolerated. Computational studies provided a rationale for the observed structure-activity relationship. Treatment of human corneal epithelial cells (HCEs) infected with HSV-1 with the hexa- and octasaccharide blocked viral induced shedding of HS which significantly reduced spread of virions. The compounds also inhibited migration and proliferation of immortalized HCEs thereby providing additional therapeutic properties.

Role of a Novel Heparanase Inhibitor on the Balance between Apoptosis and Autophagy in U87 Human Glioblastoma Cells.

BACKGROUND: Heparanase (HPSE) is an endo-ß-glucuronidase that cleaves heparan sulfate side chains, leading to the disassembly of the extracellular matrix, facilitating cell invasion and metastasis dissemination. In this research, we investigated the role of a new HPSE inhibitor, RDS 3337, in the regulation of the autophagic process and the balance between apoptosis and autophagy in U87 glioblastoma cells. METHODS: After treatment with RDS 3337, cell lysates were analyzed for autophagy and apoptosis-related proteins by Western blot. RESULTS: We observed, firstly, that LC3II expression increased in U87 cells incubated with RDS 3337, together with a significant increase of p62/SQSTM1 levels, indicating that RDS 3337 could act through the inhibition of autophagic-lysosomal flux of LC3-II, thereby leading to accumulation of lipidated LC3-II form. Conversely, the suppression of autophagic flux could activate apoptosis mechanisms, as revealed by the activation of caspase 3, the increased level of cleaved Parp1, and DNA fragmentation. CONCLUSIONS: These findings support the notion that HPSE promotes autophagy, providing evidence that RDS 3337 blocks autophagic flux. It indicates a role for HPSE inhibitors in the balance between apoptosis and autophagy in U87 human glioblastoma cells, suggesting a potential role for this new class of compounds in the control of tumor growth progression.

Pharmacological hypothesis: A recombinant probiotic for taming bacterial ß-glucuronidase in drug-induced enteropathy.

Advances in pharmacomicrobiomics have shed light on the pathophysiology of drug-induced enteropathy associated with the therapeutic use of certain non-steroidal anti-inflammatory drugs, anticancer chemotherapies and immunosuppressants. The toxicity pathway results from the post-glucuronidation release and digestive accumulation of an aglycone generated in the context of intestinal dysbiosis characterized by the expansion of ß-glucuronidase-expressing bacteria. The active aglycone could trigger direct or indirect inflammatory signaling on the gut epithelium. Therefore, taming bacterial ß-glucuronidase (GUS) activity is a druggable target for preventing drug-induced enteropathy. In face of the limitations of antibiotic strategies that can worsen intestinal dysbiosis and impair immune functions, we hereby propose the use of a recombinant probiotic capable of mimicking repressive conditions of GUS through an inducible plasmid vector.

Mechanism-based heparanase inhibitors reduce cancer metastasis in vivo.

Heparan sulfate proteoglycans (HSPGs) mediate essential interactions throughout the extracellular matrix (ECM), providing signals that regulate cellular growth and development. Altered HSPG composition during tumorigenesis strongly aids cancer progression. Heparanase (HPSE) is the principal enzyme responsible for extracellular heparan sulfate catabolism and is markedly up-regulated in aggressive cancers. HPSE overactivity degrades HSPGs within the ECM, facilitating metastatic dissemination and releasing mitogens that drive cellular proliferation. Reducing extracellular HPSE activity reduces cancer growth, but few effective inhibitors are known, and none are clinically approved. Inspired by the natural glycosidase inhibitor cyclophellitol, we developed nanomolar mechanism-based, irreversible HPSE inhibitors that are effective within physiological environments. Application of cyclophellitol-derived HPSE inhibitors reduces cancer aggression in cellulo and significantly ameliorates murine metastasis. Mechanism-based irreversible HPSE inhibition is an unexplored anticancer strategy. We demonstrate the feasibility of such compounds to control pathological HPSE-driven malignancies.

Microbial enzymes induce colitis by reactivating triclosan in the mouse gastrointestinal tract.

Emerging research supports that triclosan (TCS), an antimicrobial agent found in thousands of consumer products, exacerbates colitis and colitis-associated colorectal tumorigenesis in animal models. While the intestinal toxicities of TCS require the presence of gut microbiota, the molecular mechanisms involved have not been defined. Here we show that intestinal commensal microbes mediate metabolic activation of TCS in the colon and drive its gut toxicology. Using a range of in vitro, ex vivo, and in vivo approaches, we identify specific microbial ß-glucuronidase (GUS) enzymes involved and pinpoint molecular motifs required to metabolically activate TCS in the gut. Finally, we show that targeted inhibition of bacterial GUS enzymes abolishes the colitis-promoting effects of TCS, supporting an essential role of specific microbial proteins in TCS toxicity. Together, our results define a mechanism by which intestinal microbes contribute to the metabolic activation and gut toxicity of TCS, and highlight the importance of considering the contributions of the gut microbiota in evaluating the toxic potential of environmental chemicals.

The Heparanase Regulatory Network in Health and Disease.

The extracellular matrix (ECM) is a structural framework that has many important physiological functions which include maintaining tissue structure and integrity, serving as a barrier to invading pathogens, and acting as a reservoir for bioactive molecules. This cellular scaffold is made up of various types of macromolecules including heparan sulfate proteoglycans (HSPGs). HSPGs comprise a protein core linked to the complex glycosaminoglycan heparan sulfate (HS), the remodeling of which is important for many physiological processes such as wound healing as well as pathological processes including cancer metastasis. Turnover of HS is tightly regulated by a single enzyme capable of cleaving HS side chains: heparanase. Heparanase upregulation has been identified in many inflammatory diseases including atherosclerosis, fibrosis, and cancer, where it has been shown to play multiple roles in processes such as epithelial-mesenchymal transition, angiogenesis, and cancer metastasis. Heparanase expression and activity are tightly regulated. Understanding the regulation of heparanase and its downstream targets is attractive for the development of treatments for these diseases. This review provides a comprehensive overview of the regulators of heparanase as well as the enzyme's downstream gene and protein targets, and implications for the development of new therapeutic strategies.

Discovery of a naturally occurring broad-spectrum inhibitor against gut bacterial ß-glucuronidases from Ginkgo biloba.

Gut bacterial ß-glucuronidases (GUS) play an important role in deconjugation of various O-glucuronides, which are tightly linked with the drug-induced intestinal toxicity. Increasing evidence has indicated that inhibition of bacterial GUS could alleviate GUS-associated intestinal toxicity, but the potent and broad-spectrum inhibitors against multiple bacterial GUS have been rarely reported. This study aimed to find potent and broad-spectrum GUS inhibitors from Ginkgo biloba. It was found that amentoflavone displayed relatively strong inhibition on three GUS including CpGUS, SpasGUS and EcGUS. Further investigations demonstrated that amentoflavone could inhibit GUS-mediated PNPG hydrolysis in a dose-dependent manner with IC50 values of 2.36 µM, 2.88 µM and 3.43 µM for CpGUS, SpasGUS and EcGUS, respectively. Inhibition kinetic studies showed that amentoflavone functioned as a non-competitive inhibitor against all tested GUS with Ki values of less than 2 µM. Docking simulations indicated that amentoflavone could tightly bind on allosteric sites of three GUS mainly via hydrogen bonding interactions, and the number of hydroxyl groups of amentoflavone played crucial roles in these interactions. Collectively, our findings suggested that amentoflavone was a potent broad-spectrum inhibitor against bacterial GUS, which can be used as a promising lead compound for developing novel agents to alleviate GUS-associated intestinal toxicity.

Inhibitory Activity of 4-O-Benzoyl-3'-O-(OMethylsinapoyl) Sucrose from Polygala tenuifolia on Escherichia coliß-Glucuronidase.

Bacterial ß-glucuronidase in the intestine is involved in the conversion of 7-ethyl-10- hydroxycamptochecin glucuronide (derived from irinotecan) to 7-ethyl-10-hydroxycamptothecin, which causes intestinal bleeding and diarrhea (side effects of anti-cancer drugs). Twelve compounds (1-12) from Polygala tenuifolia were evaluated in terms of ß-glucuronidase inhibition in vitro. 4-O-Benzoyl-3'-O-(O-methylsinapoyl) sucrose (C3) was highly inhibitory at low concentrations. C3 (an uncompetitive inhibitor) exhibited a ki value of 13.4 µM; inhibitory activity increased as the substrate concentration rose. Molecular simulation revealed that C3 bound principally to the Gln158-Tyr160 enzyme loop. Thus, C3 will serve as a lead compound for development of new ß- glucuronidase inhibitors.

Discovery of a series of 5-phenyl-2-furan derivatives containing 1,3-thiazole moiety as potent Escherichia coli ß-glucuronidase inhibitors.

Gut microbial ß-glucuronidases have drawn much attention due to their role as a potential therapeutic target to alleviate some drugs or their metabolites-induced gastrointestinal toxicity. In this study, fifteen 5-phenyl-2-furan derivatives containing 1,3-thiazole moiety (1-15) were synthesized and evaluated for their inhibitory effects against Escherichia coli ß-glucuronidase (EcGUS). Twelve of them showed satisfactory inhibition against EcGUS with IC50 values ranging from 0.25 µM to 2.13 µM with compound 12 exhibited the best inhibition. Inhibition kinetics studies indicated that compound 12 (Ki = 0.14 ± 0.01 µM) was an uncompetitive inhibitor for EcGUS and molecular docking simulation further predicted the binding model and capability of compound 12 with EcGUS. A preliminary structure-inhibitory activity relationship study revealed that the heterocyclic backbone and bromine substitution of benzene may be essential for inhibition against EcGUS. The compounds have the potential to be applied in drug-induced gastrointestinal toxicity and the findings would help researchers to design and develop more effective 5-phenyl-2-furan type EcGUS inhibitors.

Heparanase inhibition preserves the endothelial glycocalyx in lung grafts and improves lung preservation and transplant outcomes.

The endothelial glycocalyx (eGC) is considered a key regulator of several mechanisms that prevent vascular injury and disease. Degradation of this macromolecular layer may be associated with post-transplant graft dysfunction. In this study, we aimed to demonstrate the benefits of eGC protection via heparanase inhibition on graft quality. We established rat models of lung grafts with damaged or preserved eGC using ischemic insult and transplanted the grafts into recipients. Lung grafts were also subjected to normothermic ex vivo lung perfusion for detailed assessment under isolated conditions. Physiologic parameters and eGC-associated cellular events were assessed in grafts before and after reperfusion. Structurally degraded eGC and highly activated heparanase were confirmed in lungs with ischemic insult. After transplant, lungs with damaged eGC exhibited impaired graft function, inflammation, edema, and inflammatory cell migration. Increased eGC shedding was evident in the lungs after reperfusion both in vivo and ex vivo. These reperfusion-related deficiencies were significantly attenuated in lungs with preserved eGC following heparanase inhibition. Our studies demonstrated that eGC plays a key role in maintaining lung graft quality and function. Heparanase inhibition may serve as a potential therapeutic to preserve eGC integrity, leading to improved post-transplant outcomes.

Cytotoxic, Cellular Antioxidant, and Antiglucuronidase Properties of the Ethanol Leaf Extract from Bulbine asphodeloides.

Bulbine asphodeloides (L.) Spreng (Xanthorrhoeaceae family), popularly known in South Africa as "ibhucu" or "Balsamkopieva," is a perennial plant traditionally used to treat skin diseases, including sunburns, rough skin, dressing burns, itches, and aging. The present study reports the cytotoxic, cellular antioxidant, and antiglucuronidase properties of the ethanol leaf extract from B. asphodeloides. The cytotoxic effect of the plant extract on human dermal fibroblast (MRHF) cells was evaluated by the bis-Benzamide H 33342 trihydrochloride/propidium iodide (Hoechst 33342/PI) dual-staining method. A validated biological cell-based assay was used to determine the cellular antioxidant activity of the extract. The antiglucuronidase and metal chelating activities were evaluated using standard in vitro methods. Lipopolysaccharide- (LPS-) induced RAW 264.7 cell model was used to determine the anti-inflammatory effect of the plant extract, and the immune-modulatory activity was performed using RAW 264.7 cells. The extract demonstrated no cytotoxic effect towards the MRHF cells at all the tested concentrations. Furthermore, the extract also possessed significant cellular antioxidant and antiglucuronidase activities, but a weak effect of metal chelating activity in a dose-dependent manner. However, the extract showed no significant anti-inflammatory and immune-stimulatory activities. Overall, the results showed that B. asphodeloides may be a useful therapeutic agent for the treatment of skin diseases, therefore supporting its ethnomedicinal usage.

Human gut bacterial ß-glucuronidase inhibition: An emerging approach to manage medication therapy.

Bacterial ß-glucuronidase enzymes (BGUSs) are at the interface of host-microbial metabolic symbiosis, playing an important role in health and disease as well as medication outcomes (efficacy or toxicity) by deconjugating a large number of endogenous and exogenous glucuronides. In recent years, BGUSs inhibition has emerged as a new approach to manage diseases and medication therapy and attracted an increasing research interest. However, a growing body of evidence underlines great genetic diversity, functional promiscuity and varied inhibition propensity of BGUSs, which have posed big challenges to identifying BGUSs involved in a specific pathophysiological or pharmacological process and developing effective inhibition. In this article, we offered a general introduction of the function, in particular the physiological, pathological and pharmacological roles, of BGUSs and their taxonomic distribution in human gut microbiota, highlighting the structural features (active sites and adjacent loop structures) that affecting the protein-substrate (inhibitor) interactions. Recent advances in BGUSs-mediated deconjugation of drugs and carcinogens and the discovery and applications of BGUS inhibitors in management of medication therapy, typically, irinotecan-induced diarrhea and non-steroidal anti-inflammatory drugs (NSAIDs)-induced enteropathy, were also reviewed. At the end, we discussed the perspectives and the challenges of tailoring BGUS inhibition towards precision medicine.

Structural Characterization and Heparanase Inhibitory Activity of Fucosylated Glycosaminoglycan from Holothuria floridana.

Unique fucosylated glycosaminoglycans (FG) have attracted increasing attention for various bioactivities. However, the precise structures of FGs usually vary in a species-specific manner. In this study, HfFG was isolated from Holothuria floridana and purified by anion exchange chromatography with the yield of ~0.9%. HfFG was composed of GlcA, GalNAc and Fuc, its molecular weight was 47.3 kDa, and the -OSO3-/-COO- molar ratio was 3.756. HfFG was depolymerized by a partial deacetylation-deaminative cleavage method to obtain the low-molecular-weight HfFG (dHfFG). Three oligosaccharide fragments (Fr-1, Fr-2, Fr-3) with different molecular weights were isolated from the dHfFG, and their structures were revealed by 1D and 2D NMR spectroscopy. HfFG should be composed of repeating trisaccharide units -{(L-FucS-α1,3-)d-GlcA-ß1,3-d-GalNAc4S6S-ß1,4-}-, in which sulfated fucose (FucS) includes Fuc2S4S, Fuc3S4S and Fuc4S residues linked to O-3 of GlcA in a ratio of 45:35:20. Furthermore, the heparanase inhibitory activities of native HfFG and oligosaccharide fragments (Fr-1, Fr-2, Fr-3) were evaluated. The native HfFG and its oligosaccharides exhibited heparanase inhibitory activities, and the activities increased with the increase of molecular weight. Additionally, structural characteristics such as sulfation patterns, the terminal structure of oligosaccharides and the presence of fucosyl branches may be important factors affecting heparanase inhibiting activity.

Entropy-driven binding of gut bacterial ß-glucuronidase inhibitors ameliorates irinotecan-induced toxicity.

Irinotecan inhibits cell proliferation and thus is used for the primary treatment of colorectal cancer. Metabolism of irinotecan involves incorporation of ß-glucuronic acid to facilitate excretion. During transit of the glucuronidated product through the gastrointestinal tract, an induced upregulation of gut microbial ß-glucuronidase (GUS) activity may cause severe diarrhea and thus force many patients to stop treatment. We herein report the development of uronic isofagomine (UIFG) derivatives that act as general, potent inhibitors of bacterial GUSs, especially those of Escherichia coli and Clostridium perfringens. The best inhibitor, C6-nonyl UIFG, is 23,300-fold more selective for E. coli GUS than for human GUS (Ki = 0.0045 and 105 µM, respectively). Structural evidence indicated that the loss of coordinated water molecules, with the consequent increase in entropy, contributes to the high affinity and selectivity for bacterial GUSs. The inhibitors also effectively reduced irinotecan-induced diarrhea in mice without damaging intestinal epithelial cells.

Oligosaccharides from fucosylated glycosaminoglycan prevent breast cancer metastasis in mice by inhibiting heparanase activity and angiogenesis.

The invasion and metastasis of tumor cells are the hallmarks of malignant diseases and the greatest obstacle to overcome. Heparanase-mediated degradation of heparan sulfate (HS) is the critical process for tumor angiogenesis and metastasis, therefore, heparanase become an attractive target for cancer research. Herein, we reported a native fucosylated glycosaminoglycan (nHG) extracted from sea cucumber Holothuria fuscopunctata and a depolymerized nHG (dHG) and its contained oligosaccharides (hs17, hs14, hs11, hs8 and hs5), acting as heparanase inhibitors. nHG and its derivatives have the ability to bind with heparanase directly, leading to significant inhibition of heparanase activity. Moreover, their apparent binding affinity to heparanase was comparable to their inhibitory effect, which was elevated along with the increase of chain length, similar to the effect of heparins. In addition, oligosaccharides inhibited the migration and invasion of 4T1 mammary carcinoma cells and human umbilical vein endothelial cells (HUVECs) and also suppressed tube formation in Matrigel matrix and angiogenesis in the chick chorioallantoic membrane (CAM) assay. In the metastatic mouse model, oligosaccharides exhibited practical antimetastatic effects on 4T1 mammary carcinoma cells. According to the reported anticoagulant activity and the low bleeding tendency of dHG and its oligosaccharides, the use of the oligosaccharides may lead to better effects on tumor patients with thrombosis tendency.

2,5-Disubstituted furan derivatives containing 1,3,4-thiadiazole moiety as potent α-glucosidase and E. coli ß-glucuronidase inhibitors.

In this paper, the 2,5-disubstituted furan derivatives containing 1,3,4-thiadiazole were synthesized and screened for their inhibitory activity against α-glucosidase and ß-glucuronidases to obtain potent α-glucosidase inhibitor 9 (IC50 = 0.186 µM) and E. coli ß-glucuronidase inhibitor 26 (IC50 = 0.082 µM), respectively. The mechanisms of the compounds were studied. The kinetic study revealed that compound 9 is a competitive inhibitor against α-glucosidase (Ki = 0.05 ± 0.003 µM) and molecular docking simulation showed several key interactions between 9 and the target including hydrogen bond and p-π stacking interaction. Derivative 26 (Ki = 0.06 ± 0.005 µM) displayed uncompetitive inhibition behavior against EcGUS. Furthermore, the result of docking revealed the furan ring of 26 may be a key moiety in obstructing the active domain of EcGUS. In addition, compound 15 exhibited significant inhibitory activity against these two enzymes, with potential therapeutic effects against diabetes and against CPT-11-induced diarrhea. At the same time, their low toxicity against normal liver tissue LO2 cells lays the foundation for in vivo studies and the development of bifunctional drug.

Amentoflavone from Selaginella tamariscina as a potent inhibitor of gut bacterial ß-glucuronidase: Inhibition kinetics and molecular dynamics stimulation.

Gut bacterial ß-glucuronidase (GUS) plays a pivotal role in the metabolism and reactivation of a vast of glucuronide conjugates of both endogenous and xenobiotic compounds in the gastrointestinal tract of human, which has been implicated in certain drug-induced gastrointestinal tract (GI) toxicity in clinic. Inhibitors of gut microbial GUS exhibited great potentials in relieving the drug-induced GI toxicity. In this study, Selaginella tamariscina and its major biflavonoid amentoflavone (AMF) were evaluated for their inhibitory activity against Escherichia coli GUS. Two selective probe substrates for GUS (a specific fluorescent probe substrate for GUS, DDAOG and a classical drug substrate for GUS, SN38G) were used in parallel for charactering the inhibition behaviors. Both the extract of S. tamariscina and its major biflavonoid AMF displayed evident inhibitory effects on GUS, and the IC50 values of AMF against GUS mediated DDAOG and SN-38G hydrolysis were 0.62 and 0.49 µM, respectively. Inhibition kinetics studies indicated that AMF showed mixed type inhibition for GUS-mediated DDAOG hydrolysis, while displayed competitive type inhibition against GUS-mediated SN-38G hydrolysis, with the Ki values of 0.24 and 1.25 µM, respectively. Molecular docking studies and molecular dynamics stimulation results clarified the role of amino acid residues Leu361, Ile363, and Glu413 in the inhibition of AMF on GUS. These results provided some foundations for the potential clinical utility of S. tamariscina and its major biflavonoid AMF for treating drug-induced enteropathy.

Inducing new bioactive metabolites production from coculture of Pestalotiopsis sp. and Penicillium bialowiezense.

Coculturing two or more fungi is a useful strategy to awaken the silent genes to produce structurally diverse and bioactive natural products. Through the coculture of Pestalotiopsis sp. and Penicillium bialowiezense, six new isoprenylated chromane derivatives, including two pairs of enantiomeric ones (1a/1b-2a/2b) and two optical pure ones (3-4), two new isoprenylated phenol glucoside derivatives (6-7), as well as eight known structural analogues (5 and 8-14), were obtained. The structures of these new compounds were characterized by NMR spectroscopy, single-crystal X-ray crystallography, and ECD calculation. The Δ10,11 double bond of pestaloficin D (5) was revised to E-configurated based on the extensive spectroscopic analyses. Compounds 1a/1b and 2a/2b were the first examples of enantiomeric isoprenylated chromane derivatives, which were successfully separated by chiral HPLC. Additionally, all the isolated compounds were evaluated for the in vitro ß-glucuronidase (GUS) and butyrylcholinesterase (BChE) inhibitory activities. Compounds 1a and 1b showed significant ß-glucuronidase inhibitory potency with IC50 values of 7.6 and 10.3 µM, respectively. Compound 14 exhibited moderate BChE inhibitory activity with an IC50 value of 21.3 µM. In addition, the structure-enzyme inhibitory activity relationship of compounds 1-14 is discussed.